The Genotoxic and Cytotoxic Effects of Bisphenol-A (BPA) in MCF-7 Cell Line and Amniocytes.

Bisphenol-A (BPA) is an industrial xenoestrogen used widely in our living environment. Recently, several studies suggested that BPA has destructive effects on DNA and chromosomes in normal body cells via estrogen receptors (ER). Therefore, BPA could be considered as an important mediator in many diseases such as cancer. However, there are still many controversial issues which need clarification. In this study, we investigated the BPA-induced chromosomal damages in MCF-7 cell line, ER-positive and negative amniocyte cells. Cytotoxicity and genotoxicity effects of BPA were also compared between these three cell groups. Expression of estrogen receptors was determined using immunocytochemistry technique. The cell cytotoxicity of BPA was measured by MTT assay. Classic cytogenetic technique was carried out for the investigation of chromosome damage. BPA, in addition to cytotoxicity, had remarkable genotoxicity at concentrations close to the traceable levels in tissues or biological fluids. Although some differences were observed in the amount of damages between ER-positive and negative fetal cells, interestingly, these differences were not significant. The present study showed that BPA could lead to chromosomal aberrations in both ER-dependent and independent pathways at some concentrations or in cell types yet not reported. Also, BPA could probably be considered as a facilitator for some predisposed cells to be cancerous by raising the chromosome instability levels. Finally, estrogen receptor seems to have a different role in cytotoxicity and genotoxicity effects.

Genomic instability and chromosomal abnormalities are well-known common features of cancer (16)(17).
Recent studies have strongly suggested that DNA damage induced by xenoestrogens and estrogen is dependent on estrogen receptors (ERs) (3,(18)(19). An in vitro study has indicated the effect of estradiol on radiation-induced chromosome aberrations in human peripheral lymphocytes (20).
BPA is considered as an estrogenic endocrine disrupting chemical which exhibits estrogen-like activity (21). BPA binds to ERs that could promote breast cancer (22).
To date many studies have indicated controversial issues concerning chromosomal aberrations induced by BPA. Although some studies suggested that BPA cannot have a genotoxic effect, some others suggested that BPA exposure can lead to chromosomal abnormalities such as aneuploidy through disruption of meiotic process (23)(24) and also genomic structural aberrations like DNA breakage (25). Recent studies have demonstrated that BPA impairs the double-strand break repair machinery in the germline and causes chromosome abnormalities (26). Furthermore, BPA induces synaptic defects, such as end-to-end chromosome associations and asynapsis (27

Confirmation of the existence of estrogen receptors in MCF-7 and female amniocytes
In order to evaluate the presence of estrogen receptors, ICC was used for all three cell groups.
For this purpose, MCF-7 was used as a positive control which clearly expressed ERs (Figure 1-A).
As it was expected, results showed that unlike the male amniocytes, the females expressed ER ( Figure   1-     As it is obvious in figure 4, the number of abnormal cells and also structural aberrations decreased in higher doses. For example no analyzable female amniocyte metaphase was found at doses 40 and 100 µg/ml which could be due to cytotoxic effects of BPA and cell arrest at these doses.

Chromosomal rearrangements
The karyotypes of the chromosomal spreads in MCF-7 cells and amniocytes were normal using GTG banding technique.

Numerical aberrations
Although, the numerical variations in BPA exposed MCF-7 cells were completely observable, but it didn't follow any particular pattern of chromosomes number variation after exposure to different concentrations of BPA (Table 2).  Because of low quality of plastics industry as well as lack of efficient recycling system in these countries, it is expected that in different populations of these countries, BPA levels should be higher than in the developed ones. The same applies to workers of plastics industry and other highly exposed people. On the other hand, many types of xenoestrogens exist in human living environments and the cumulative effects of these components in in vivo situation, may have the same effects as the examined toxic levels of BPA in the present in vitro investigation.